Abstract The mechanisms by which ischemic stroke induces brain cell death are complex. Within seconds to minutes after ischemia, a series of cellular reactions, collectively called the ischemic cascade, are induced that markedly increase brain cell death and expand the ischemic zone. Approximately 60-90 min after ischemia, a second wave of reactions occurs with the potential to kill even more brain cells than the initial ischemic event. This second wave, referred to as secondary brain injury, is dominated by neutrophil recruitment, activation, and release of myeloperoxidase (MPO). Recently, we reported that N-acetyl-Lysyltyrosylcysteine amide (KYC) effectively reduced brain lesions by >56% and improved neurological severity scores in mice subjected to middle cerebral artery occlusion (MCAO) by >60%. Although KYC is highly effective at preserving brain and neurological function after focal ischemia, it has little to no effects on reducing damage induced by the ischemic cascade. To protect brain cells from the ischemic cascade we propose to design and develop trifunctional tripeptides that will 1) bind intracellular free Ca2+ [Ca2+]i, 2) scavenge free radicals and oxidants, and 3) shuttle oxidants into the GSH system and be regenerated to active monomers in the same way as KYC. In this R21 application we propose two aims. Aim 1: (A) Characterize the tripeptides with respect to their ability to bind Ca2+ and scavenge free radicals and oxidants. (B) Characterize the tripeptides with respect to their ability to reduce [Ca2+]i and scavenge free radicals and oxidants in HT22 cell cultures. Aim 2: Determine the effects of tripeptides on (A) glutamate and oxygen-glucose-deprivation (OGD) induced brain cell death and (B) brain infarct size, oxidative injury and neurological deficits in MCAO rats in long term and time window studies. Successful completion of these studies will allow us to conduct combination studies with KYC to determine if targeting both the ischemic cascade and secondary brain injury provide better outcomes than either pathway alone. Findings from this R21 may have a significant impact on the treatment and care of people who have suffered ischemic stroke or other forms of ischemic or traumatic brain injury.